Protective Effect Of Zerumbone On CCl₄-induced Acute Liver Injury In Mice And Preliminary Mechanism Study

Objective:This study amied to investigate the effect of ZER against acute liver injury in CCl₄-induced mice model and to explore the underlying mechanism.Methods:After a five-day acclimation based on body weight,sixty ICR mice were divided into six groups of 10 animals each,as follows:The control（Con）group,the CCl₄model group,the ZER-L(1.25?mol·kg^-1 of body weight（BW）,dissolved in DMSO)group,the ZER-M(5?mol·kg^-1)group,the ZER-H(20?mol·kg^-1)group,and the positive control group treated with BIF(350?mol·kg^-1).The final concentration（v/v）of DMSO in the in vivo experiment was 0.1%.During the experiment,the mice were given a standard diet.All mice were intraperitoneally injected with corresponding drugs for 5 consecutive days:The control group and the CCl₄ model group injected with DMSO,three ZER groups with ZER with the corresponding concentrations,and the positive control group with BIF.Two hours after the last administration,all the animals from each group received a one-time intraperitoneal injection of CCl₄（0.01m L/g body weight,0.2%in soybean oil）to establish the ALI model,except for the control group,which were received the same volume of soybean oil.Twelve hours fasting followed with the CCl₄ challenge.The mice were sacrificed 12 h after the CCl₄challenge.Blood and liver tissue were collected for experiments.The serum was obtained by centrifuging and then immediately subjected to the following biochemical analysis.For the liver,a whole lobe without any mechanical damage was fixed in 10%formalin for histopathological analysis,while the rest of the liver tissue samples were stored in liquid nitrogen for further experiments.The activities of glutamic-pyruvic transaminase（ALT）and glutamic oxalacetic transaminase（AST）in serum and the activities or levels of superoxide dismutase（SOD）,glutathion peroxidase（GSH-Px）,glutathione（GSH）,and malondialdehyde（MDA）in liver homogenates were determined by commercial kits according to the manufacturer’s instructions.The liver lobes fixed in 10%formalin were embedded in paraffin and processed into 4μm thick sections,which were subjected to H&E staining for histopathological examination.The levels of interleukin-6（IL-6）and Tumor necrosis factor–α（TNF-α）both in serum and in liver tissue were analyzed by ELISA kits according to the manufacturer’s instructions.Western Blot was used to detect the expressions of TLR4,NF-κB and COX-2 proteins in mouse liver.The cytotoxicity of ZER and LPS toward RAW246.7 cells were evaluated by MMT assays.ELISA was used to detect the effect of LPS on the secretion of IL-6 and TNF-αin RAW264.7 cell.Establishing an inflammatory cellular model by LPS stimulated RAW264.7 to investigate whether ZER plays its role in liver protection by alleviating inflammation.And the expression of TLR4,NF-κB and COX-2 proteins were detected by Western Blot.Results:（1）.Results of serum AST and ALT in CCl₄-induced mice showed that the AST and ALT levels of mice in the model group were significantly increased（P<0.01）compared with the control group.Which were significantly decreased in a dose-dependent manner in the ZER-L,ZER-M and ZER-H groups,indicating that ZER had a protective effect on acute liver injury caused by CCl₄.（2）.Results of liver index showed that ZER could reduce the increased level of liver index caused by CCl4 in mice in a dose-dependent manner.（3）.HE results showed that the liver tissue structure of the control group was normal,and which was significantly damaged in CCl₄ group,there were moderate hepatocyte necrosis in the central lobular area.After ZER pretreatment,the tissue structure gradually tended to be normal,the liver cells were arranged neatly,and the liver cell structure tended to be complete.The area of liver tissue necrosis decreased from 49%in the model group to 39.4%（ZER-L）,26.9%（ZER-M）and 19.7%（ZER-H）.Serum test and histopathological analysis confirmed the liver protective effect of ZER.（4）.Biochemical analysis results indicated that ZER pretreatment restored the enzyme activity of SOD and GSH-Px,restored the accumulation of GSH,and reduced the generation of MDA in a dose-dependent manner.The results of the detection of antioxidant enzymes and lipid peroxidation in tissues preliminarily revealed that one possible mechanism of ZER against CCl₄-induced ALI was related to the oxidative stress pathway.（5）.Results of ELISA displayed that ZER intraperitoneal injection could reduce the release of IL-6 and TNF-α.These results suggest that another possible mechanism of ZER against CCl₄-induced ALI may related to the inflammatory response pathway.（6）.Western Blot results revealed that ZER inhibited the expression of TLR4,NF-κB p-p65 and COX-2 proteins in the liver tissues of mice with acute liver injury caused by CCl₄.（7）.Results of MTT and ELISA suggested that the optimal concentration of LPS for the establishment of inflammatory cellular model in RAW264.7 cell was 1?g·m L^-1.And determined two ZER treatment groups:2.5?mol·L^-1 as the low dose group（ZER-L）and 5?mol·L^-1 as the high dose group（ZER-H）.Western Blot results showed that ZER significantly reduced the protein expression levels of TLR4,NF-κB p-p65 and COX-2.Conclusions:（1）.ZER has a protective effect on acute liver injury induced by CCl₄ in mice.（2）.ZER may play a protective role in acute liver injury by reducing oxidative stress and fightting inflammation.（3）.ZER plays its role in liver protection by alleviating the inflammatory response through the TLR4/NF-κB/COX-2 signaling pathway.